Exogenous Angiotensin-(1–7) Provides Protection Against Inflammatory Bone Resorption and Osteoclastogenesis by Inhibition of TNF-α Expression in Macrophages

Renin–angiotensin–aldosterone system plays a crucial role in the regulation of blood pressure and fluid homeostasis. It is reported to be involved in mediating osteoclastogenesis and bone loss in diseases of inflammatory bone resorption such as osteoporosis. Angiotensin-(1–7), a product of Angiotensin I and II (Ang I, II), is cleaved by Angiotensin-converting enzyme 2 and then binds to Mas receptor to counteract inflammatory effects produced by Ang II. However, the mechanism by which Ang-(1–7) reduces bone resorption remains unclear. Therefore, we aim to elucidate the effects of Ang-(1–7) on lipopolysaccharide (LPS)-induced osteoclastogenesis. In vivo, mice were supracalvarial injected with Ang-(1–7) or LPS ± Ang-(1–7) subcutaneously. Bone resorption and osteoclast formation were compared using micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) stain, and real-time PCR. We found that Ang-(1–7) attenuated tumor necrosis factor (TNF)-α, TRAP, and Cathepsin K expression from calvaria and decreased osteoclast number along with bone resorption at the suture mesenchyme. In vitro, RANKL/TNF-α ± Ang-(1–7) was added to cultures of bone marrow-derived macrophages (BMMs) and osteoclast formation was measured via TRAP staining. The effect of Ang-(1–7) on LPS-induced osteoblasts RANKL expression and peritoneal macrophages TNF-α expression was also investigated. The effect of Ang-(1–7) on the MAPK and NF-κB pathway was studied by Western blotting. As a result, Ang-(1–7) reduced LPS-stimulated macrophages TNF-α expression and inhibited the MAPK and NF-κB pathway activation. However, Ang-(1–7) did not affect osteoclastogenesis induced by RANKL/TNF-α nor reduce osteoblasts RANKL expression in vitro. In conclusion, Ang-(1–7) alleviated LPS-induced osteoclastogenesis and bone resorption in vivo via inhibiting TNF-α expression in macrophages. Supplementary Information The online version contains supplementary material available at 10.1007/s00223-024-01257-6.


Introduction
The renin-angiotensin-aldosterone system (RAAS) is a critical hormonal system that is involved in the pathogenesis of conditions such as hypertension, diabetic kidney disease, and lung injury [1][2][3].RAAS is mainly composed of renin, Angiotensin (Ang) I, Ang II, other downstream peptide hormones, and angiotensin-converting enzymes (ACE).Renin converts angiotensinogen into Ang I, which is subsequently transformed into the biologically active octapeptide known as Ang II by ACE.As a crucial factor in hypertension and a prime target for therapeutic interventions, Ang II induces vasoconstriction through Ang II type 1 receptors (AT1R), which is counteracted by AT2R [4].More recently, further elements of the RAAS have been identified, with Ang-(1-7) emerging as one of the most extensively studied among them [5,6].Ang-(1-7) is a heptapeptide produced through the cleavage of Ang II by ACE2.It binds to its unique Mas receptor (MasR), counteracting the effects triggered by Ang II, including vasoconstriction, inflammation, and insulin resistance [7][8][9][10][11].
Osteoclasts are the cells responsible for bone resorption and osteoclastogenesis is the process through which bone marrow cells differentiate into osteoclasts after being exposed to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL) [17,18].M-CSF and RANKL are produced by various cell types, including osteoblasts [19].M-CSF binds to CSF-1 receptor on macrophages that initiate subsequent intercellular communication leading to osteoclast differentiation [20].RANKL binds to its receptor (RANK) on the surface of osteoclast precursors, driving their differentiation into mature osteoclasts.OPG is a soluble decoy receptor for RANKL and prevents its interaction with RANKL reducing osteoclast formation [21].Tumor necrosis factor (TNF)-α promotes osteoclastogenesis by inducing the expression of M-CSF and RANKL, while also independently promoting osteoclast maturation in vitro [22,23] and in vivo [24,25].Lipopolysaccharide (LPS) triggers the release of TNF-α by macrophages leading to osteoclast formation [26].

Histological Analysis
Mice were divided into four injection groups: 1 × phosphatebuffered saline (PBS), LPS (100 μg/day), LPS + Ang-(1-7) , and Ang-(1-7) (100 μg/day) only.Each group contains at least 4 samples.The mice received a subcutaneous supracalvarial injection according to the assigned group for five consecutive days.On the sixth day, mice were sacrificed by cervical dislocation after anesthetized by isoflurane inhalation (Pfizer, New York, NY).The calvariae were dissected and fixed in 10% formalin for 3 days at 4 ℃.Micro-computed tomography (Micro-CT) scanning using ScanXmate (Comscan, Kanagawa, Japan) and 3-dimensional reconstruction for comparing the destructed areas by TRI/3D-Bon (RATOC System Engineering, Tokyo, Japan) had been conducted after fixation.Photoshop (Adobe, San Jose, CA) is used for specifying the absorption area in the calvariae images, while ImageJ (NIH, Bethesda, MD) is used to calculate the proportion of this area in a specified area.Then decalcification in 14% ethylenediaminetetraacetic acid (EDTA) was processed at room temperature for 1 week.The sagittal sutures of calvariae were divided into three equal parts from the coronal plane and subjected to a dehydration in a tissue processer (TP1020, Leica, Wetzlar, Germany).Dehydrated bone pieces were embedded in paraffin and cut into 5-µm sections perpendicular to the sagittal suture with a microtome (Leica).Sections were stained with tartrate-resistant acid phosphatase (TRAP) solution.To make the TRAP solution: Fast Red Violet, Naphthol AS-MX phosphate and N, N′-Dimethylformamide were incubated in a mixture of 0.5 M sodium tartrate dihydrate and 0.1 M sodium acetate (pH 5.0).The nucleus was counterstained with hematoxylin.All observation and picturing were performed with a digital camera DP-72-SET-B (OLYMPUS, Tokyo, Japan) and cellSens Standard (OLYMPUS).TRAP-positive multinucleated cells (with ≥ three nuclei) closely adjacent to the red-stained bone wall in the sagittal suture mesenchyme were counted manually (Supplementary Fig. 1).Statistical analysis was based on an average of 10 sections from one sample [28].

Isolation and Culture of Peritoneal Macrophages
Mice were euthanized and the peritoneum was exposed without breaking the serous membrane.A 21G needle and syringes were used to inject 6 mL ice-cold sterilized 1 × PBS beneath the peritoneal viscera over the inner side of the femur.Generally, 5 mL peritoneal lavage fluid was collected.Cells were harvested by centrifuging (1500 rpm, 8 min, 4 ℃) in 1 × PBS.Filtered cells were seeded in 12-well plates, and non-adhesive cells were washed two hours later.Then adherent cells were incubated in 37 ℃ overnight and cultured with LPS ± Ang-(1-7) for 3 days to study the effect of Ang-(1-7) on macrophages TNF-α expression.

RNA Extraction and Real-Time Reverse Transcription-Polymerase Chain Reaction
Calvariae tissues were homogenized by stirring with ceramic beads in TRIzol reagent (Ambion: Thermo Fisher Scientific) using Micro-Smash MS-100R (TOMY SEIKO, Tokyo, Japan).Chloroform (Wako, Osaka, Japan) was added to the mixture at one-fifth volume of the TRIzol and centrifuged (14,000 rpm, 10 min, 4 ℃) to collect the supernatant containing RNA.RNA was then extracted following manufacturer's instruction using RNeasy Kits (QIAGEN, Hilden, Germany).The cells were lysed with a 100:1 mixture of RLT and 2-mercaptoethanol and then transferred to the QIAShredder for homogenization followed by centrifugation (14000 rpm, 2 min, 4 ℃).Total RNA was dissolved in RNase-free water and stored at − 80 ℃.RNA concentration and quality was checked using a Nano-Photometer (Implen, Munich, Germany).cDNA was made by Super-script IV Reverse Transcriptase system using Oligo dT primer (Invitrogen, Thermo Fisher Scientific).Primers utilized in realtime PCR are listed in Table 1.GAPDH was selected as an internal control gene, and TB Green® Premix Ex Taq™ II was used (TaKaRa, Shiga, Japan).

Western Blot
Peritoneal macrophages stimulated by LPS ± Ang-(1-7) for 0, 5, 15, and 30 min were lysed in radioimmunoprecipitation assay buffer consisting of 1% protease and phosphatase inhibitor after 3 h of starvation in serum-free α-MEM.QIAShredder was used to remove cell pellets (14,000 rpm, 2 min, 4 ℃) and homogenize total protein [30].Protein concentration was quantified with a BCA assay kit (Thermos fisher Scientific) and a microplate reader was used to measure the absorbance at 595 nm.Protein was stored in -80℃ or mixed with sample buffer and denatured at 95 ℃ for 5 min.The sample buffer was prepared by combining 9 parts of 4 × Laemmli with 1 part of 2-mercaptoethanol, and each protein sample was mixed with the sample buffer in a 3:1 volume ratio.Denatured protein samples were stored in -20 ℃.
Protein was loaded on Precast Gels (Biorad, Hercules, CA) in 1 × Tris/Glycine/SDS buffer (120 V,1 h) and transferred to the PVDF membrane by the Trans-Blot Turbo Transfer System (Biorad).The membrane was blocked with a mixture of 4 g block ace powder and 20 mg sodium azide dissolved in 100 mL 1 × Tris -buffered saline with 1% Triton X-100 (TBS-T).Primary and secondary antibodies (Table 2) were diluted in immunoreaction enhancer solution (TOYOBO, Osaka, Japan) in specific dilutions.Membranes were washed in 1 × TBS-T for 10 min twice and 1 × TBS once prior to and after secondary antibody incubation.Membrane reacted with West Femto Maximum Sensitivity Substrate was detected by FUSION FX, captured by Evolution-capt Edge (VILBER, Collégien, France), and analyzed by ImageJ using a inserted band quantification Macro [31].

Statistical Analysis
All data are expressed as the mean ± standard deviation.Scheffe's test and paired t-test were used to assess the differences between groups.Statistical significance was defined as p < 0.05.Scheffe's test is a one-way ANOVA post hoc test, and it corrects alpha for complex mean comparisons with a narrower confidential interval.Data were analyzed using the Excel statistics Statcel 3 (Microsoft, Seattle, WA).

Ang-(1-7) Suppressed LPS-Induced Calvarial Bone Resorption and In Vivo Osteoclastogenesis
We dissected the injected calvariae to analyze the effect of Ang-(1-7) on bone resorption in vivo.We elevated the bone-resorbed area by Micro-CT scanning and found that LPS distinctly destructed the bone structure around the anterior suture's junction.LPS + Ang-(1-7) reduced the extent of the destruction.Ang-(1-7) alone had no effect on the normal structure of calvaria (Fig. 1A, B).According to histological analysis of calvariae sections stained with TRAP solution, LPS stimulation increased the number of TRAP-positive multinucleated cells, while the addition of Ang-(1-7) decreased it (Fig. 1C, D).We also evaluated TNF-α, RANKL, TRAP, and Cathepsin K (CTSK) expression levels using excised calvarial tissue by real-time PCR.As a result, LPS significantly elevated the mRNA expression level of TNF-α, RANKL, TRAP, and CtsK.In comparison with this, LPS + Ang-(1-7) inhibited these inflammatory or osteoclastogenic biomarkers production responding to LPS (Fig. 2).

Ang-(1-7) Does Not Affect RANKL Expression of Osteoblasts
We cultured osteoblasts to study if Ang-(1-7) affects LPSinduced RANKL expression and osteoclast formation.Isolated primary osteoblasts were cultured with LPS for the specific period (0, 3, 6, 12 h) to evaluate the time required for LPS to induce RANKL expression.LPS induced RANKL expression after 3 h (Fig. 4A).Therefore, 3 h and 48 h were selected to investigate the effect of Ang-(1-7) on short-and long-term stimulation of LPS on RANKL production.LPS significantly upregulated RANKL expression and downregulated OPG expression, increasing the ratio of RANKL/OPG in both 3-and 48-h cultures of LPS ± Ang-(1-7) (Fig. 4B-G).However, Ang-(1-7) had no effect on LPS-induced RANKL or OPG expression.

Discussion
The ACE 2/Ang-(1-7)/MasR axis has gained significant attention over the past decades due to its antagonistic role in relation to Ang II.The immunomodulatory property of RAAS emerged in the fields of oncology, transplantation, and intensive care medicine indicating its extensive interaction with inflammatory regulation [32].Moreover, the localized expression of RAAS components in bone has been linked to various bone-destructive conditions, including but not limited to osteoporosis and periodontitis [12,33].Ang-(1-7) has been observed to ameliorate the deterioration of osteomicrostructure in an OVX-rat model [13].The underlying mechanism is believed to involve anti-inflammatory and anti-osteoclastogenic properties.Given that inflammation is widely regarded as a contributor to bone degradation and that Ang-(1-7) is anti-inflammatory, our study investigated the effects of the bioactive heptapeptide Ang-(1-7) on LPS-induced inflammatory osteoclast formation and bone destruction.
Ang-(1-7) inhibits inflammation in vivo, possibly by reducing serum levels of pro-inflammatory cytokines such as TNF-α and IL-6 [34].TNF-α is a pivotal pro-inflammatory factor released by macrophages in innate immune responses upon stimulation by LPS [35].Given that LPS is capable of promoting inflammatory osteoclastogenesis [26], our initial focus was on investigating whether Ang-(1-7) inhibits LPS-induced bone resorption and osteoclast formation.Calvaria subcutaneously injection was chosen based on previous study and its specialty in inducing local inflammation [29].Consequently, supracalvarial injection of LPS leads to increased osteoclast numbers and bone resorption around the suture mesenchyme which decreased in Ang-(1-7) + LPS injection compared to LPS injection alone.In vivo mRNA expression levels of TNF-α, RANKL, TRAP, and CTSK were in line with the results obtained from RANKL, originating from a diversity of cells including osteoblasts [19], is a potent inducer of osteoclast differentiation by binding to its receptor RANK on osteoclast precursors and pre-osteoclasts.Also, TNF-α can increase osteoclast formation as TNF receptors knockout mice experienced suppressed osteoclastogenesis reported in a previous study [36].Its ability to induce hyperosteoclastogenesis independent of RANKL is still controversial, as Lam and colleagues stated that TNF-α cannot induce any formation of osteoclast without the permission of RANKL [37].In this case, a micro-amount of RANKL or osteoblast/stromal cell contaminated the serum or our cultures may explain the hyperosteoclastogenic phenomenon.Considering that TNF-α and RANKL are central to osteoclastogenesis, we sought to investigate the effects of Ang-(1-7) on osteoclast formation induced by these two factors in vitro.To accomplish this, we cultured bone marrow cells with M-CSF, which serve as osteoclast precursors.The measurement result of TRAP-positive multinucleated osteoclasts suggested that Ang-(1-7) does not inhibit osteoclastogenesis by directly acting on osteoclast precursors.This finding contrasts with the previous studies [35], where 10 −7 M (100 nM) Ang-(1-7) inhibited osteoclastogenesis.Conversely, both 10 −7 M and 10 −5 M (100 μM) concentrations of Ang-(1-7) exhibited no inhibitory effects in our study.Although we isolated bone marrow cells from long bones, the differences in the animal model (rat or mouse), incubation period with M-CSF (1-10 days), and M-CSF concentration (10-100 ng/ mL), cell culture conditions (administration of regents such as daily treat with Ang-(1-7) or extra stimulation of LPS, and stimulation period (5-10 days)) may have gave rise to the opposing effects of Ang(1-7) in the three different studies [12,38].
Accordingly, investigating the upstream regulatory factors becomes imperative.As described earlier, RANKL is the essential molecule facilitating communication between osteoblast and osteoclast.RANKL is tightly linked to osteoclast activation and alveolar bone loss in periodontitis [39].A previous study demonstrated that LPS boosted RANKL expression in bone marrow stromal cells [40].In a different context, Ang-(1-7) decreased RANKL expression in ovariectomized rats after 6 weeks of treatment [13].Building on the evidence presented before, an intuitive experiment focusing on the effect of Ang-(1-7) on osteoblasts RANKL expression is necessary.We isolated neonatal calvarial osteoblasts and cultured them with LPS for 3, 6, or 12 h to identify the time point at which RANKL expression increases.RANKL expression increased at 3 h.Therefore, we cultured osteoblasts with LPS ± Ang-(1-7) for 3 h to assess the effect of Ang-(1-7) on LPS-stimulated RANKL expression.In addition, we conducted an identical 48-h experiment to explore long-term culture outcome.Nonetheless, Ang-(1-7) did not exhibit any effect on LPS-induced osteoblast RANKL expression in vitro.
As a pro-inflammatory cytokine substantially released from macrophage upon LPS stimulation, TNF-α is responsible for osteoclastogenesis not only through direct induction [24] but also by stimulating osteoblasts to express RANKL [41].Ang-(1-7) has been reported to eliminate TNF-α expression from peritoneal macrophage [42].Given that TNF-α promotes inflammatory bone resorption, we investigated Ang-(1-7)'s effect on LPS-induced TNF-α expression from peritoneal macrophages.In line with previous research and our in vivo study, Ang-(1-7) decreased TNF-α expression in peritoneal macrophages after 72 h of culture.Taking into account that TNF-α could accelerate osteoclastogenesis through both direct and indirect pathways, our study indicates that Ang-(1-7) suppresses LPS-induced osteoclast formation by inhibiting TNF-α production in macrophages.
The MAPK pathway modulates various cellular activities, including proliferation, differentiate, and survival.It is activated during inflammation in response to diverse cellular stresses [43].The phosphorylation of key proteins ERK1/2, p38, and JNK in each pathway leads to the secretion of cytokines and chemokines in macrophages induced by LPS [44].The NF-κB pathway is crucial to modulate inflammatory response toward various stimuli.Toll-like receptors, upon stimulation by LPS, transduce cellular signals and mediate IκBα phosphorylation downstream, thereby activating the classical NF-κB pathway.This activation prompts the cell to produce multiple factors that combat inflammation [45].Therefore, we studied the activation of MAPK and NF-κB pathways by culturing peritoneal macrophages with LPS ± Ang-(1-7) for specific periods (0, 5, 15, 30 min) after 3 h starvation to exclude the influence of FBS.Through Western blotting analysis, all three phosphorylated proteins in MAPK pathway exhibited a rapid increase at 5 or 15 min activated by LPS, followed by a declining trend after 15 min.Among these, phosphorylation of p38 and ERK1/2 initiated by LPS was significantly reduced by Ang-(1-7), while p-JNK was not influenced by Ang-(1-7).Phosphorylated IκB showed a gentle rise until 30 min, and significant inhibition of Ang-(1-7) was observed at 15 min.The effect of Ang-(1-7) on the MAPK and NF-κB pathway activation may be responsible for reduced TNF-α expression in macrophages.
However, there may be some possible limitations in this study.To fully understand the effects of Ang-(1-7), a more systemic analysis is required, as the current animal model only involves localized inflammation.Additionally, exploring the effects of ACE2 and Mas receptor in conjunction with Ang-(1-7) would provide a more comprehensive understanding of RAAS.Addressing these limitations in future studies would result in a more accurate understanding of the effects of Ang-(1-7) and its potential as a therapeutic agent.
Ang-(1-7) is underrepresented in clinical studies despite its potential role in alleviating pathological conditions closely associated with Ang II, such as cardiovascular and renal diseases.Given its significant involvement in inflammatory mediation, particularly in inflammatory bone lesions, there is an urgent need for further investigation in the context of clinical trials targeting skeletal degenerative diseases under inflammation like periodontitis, osteoporosis, and rheumatic arthritis.
In summary, inflammation is interconnected with numerous chronic diseases, such as atherosclerosis, diabetes, and arthritis.ACE2/Ang-(1-7)/MasR axis possesses anti-inflammatory properties and holds potential benefits for bonerelated conditions like osteoporosis and periodontitis.Our study has demonstrated that Ang-(1-7) is anti-inflammatory and reduces bone resorption in vivo.More importantly, we In summary, our study showed that Ang-(1-7) alleviated LPS-induced osteoclastogenesis and bone resorption via the inhibition of TNF-α expression in vivo without inhibiting osteoclastogenesis directly (Fig. 6).This process may be caused by inhibiting the intracellular activation of MAPK pathway and NF-κB pathway in macrophages leading to TNF-α expression inhibition.

Fig. 1
Fig.1Ang-(1-7) suppressed LPS-induced calvarial bone resorption and osteoclast formation in vivo.A Three-dimensional reconstructed calvariae.The red area showed on the top left refers to the destructed area around the anteriorfontanelle suture, and pixels were automatically selected using Photoshop; B Percentage of the destructed bone area in a specific-sized square (50 × 70 pixels) was analyzed by ImageJ; C TRAP staining of sagittal suture mesenchyme; D Number of TRAP-positive cells.Data for analysis are average number of over ten valid sections from every sample.Scheffe's test was used to determine the statistical significance of differences between groups (n = 4-5; **p < 0.01)

Fig. 2
Fig. 2 Ang-(1-7) decreased the LPS-elevated inflammatory and osteoclastic biomarkers in vivo analyzed using real-time PCR.Data are shown in the form of folds relative to the PBS group which has been standardized into 1.A TNF-α mRNA expression level relative to GAPDH; B RANKL mRNA expression level relative to GAPDH; C TRAP mRNA expression level relative to GAPDH; D CTSK mRNA expression level relative to GAPDH.Non-significant data have not been labeled.Scheffe's test was used to determine the statistical significance of differences between groups (n = 4; **p < 0.01 *p < 0.05)

Fig. 3
Fig. 3 Ang-(1-7) does not affect RANKL or TNF-αinduced osteoclastogenesis.Bone marrow cells culture with M-CSF for 3 days were used as BMMs.A RANKL-induced osteoclastogenesis under a light scope; B TNF-α-induced osteoclastogenesis.C, D Manually counted number of TRAP-positive multinucleated cells; The scale bar is 200 μm.Scheffe's test was used to determine the statistical significance of differences between groups (n = 4; **p < 0.01)

Fig. 5 Fig. 6
Fig. 5 Ang-(1-7) suppressed LPS-induced TNF-α releasing and the MAPKs and NF-κB pathway activation in peritoneal macrophages.A Real-time PCR result of TNF-α mRNA expression level relative to GAPDH for 3 days culture (n = 4, *p < 0.05; **p < 0.01).B, C Western blotting for effect of Ang-(1-7) on phosphorylated proteins in relation to non-phosphorylated proteins and β-actin in the MAPKs and NF-κB pathway D bands quantification of each phosphorylated protein; Scheffe's test and paired t-test were used to determine the statistical significance of differences between groups in realtime PCR and Western blotting, respectively.Non-significant data have not been labeled.(n = 3 for WB; #p < 0.05)

Table 2
Antibodies and specific dilutions used for Western blotting